One of the major obstacles to the efficacy of cancer therapy is the tumor microenvironment that often outgrows its blood supply and harbors hypoxic regions that are resistant to chemo- and radiotherapy. TRAIL is part of the host immune system that suppresses cancer and its metastases and both TRAIL as well as TRAIL receptor agonist antibodies are currently in early phase clinical trial testing. In preliminary studies we have uncovered evidence that certain tumor cells, including p53-null human HCT116 colon tumor cells that are sensitive to TRAIL under normoxic conditions, become significantly resistant to TRAIL under severely hypoxic conditions. Such tumor cells are not effectively sensitized to TRAIL under hypoxia by combination therapies that include TRAIL plus 5- Fluorouracil, TRAIL plus irinotecan, or TRAIL plus the hypoxia sensitizer Tirapazamine. This prompted us to develop and carry out a high throughput chemical library screen that has identified a number of small molecules, including a family of structurally-related nucleoside analogues we refer to as the SLMs. SLMs are potent sensitizers of TRAIL under hypoxia and some family members have cytotoxic effects as single agents under hypoxia. In vivo studies reveal anti-tumor effects of novel small molecules combined with TRAIL and novel non-invasive whole animal optical imaging reveals that such combinations are associated with reduced levels of hypoxia within treated tumors as well as reduced vascularity. These are exciting findings that merit further investigation through the following approaches: Specific Aim #1: Investigate mechanisms of resistance to TRAIL therapy under hypoxia with specific focus on c-Myc, Mcl-1, HIF and NFkB signaling pathways. Specific Aim #2: Investigate mechanisms of sensitization to TRAIL under hypoxia by novel small molecules, SLMs, isolated from high throughput chemical library screening. Specific Aim #3: Investigate the impact of the tumor microenvironment on TRAIL plus SLM sensitivity through non-invasive in vivo imaging of tumor hypoxia and anti-tumor effects. This proposal is highly relevant to the understanding of the barriers to effective anti-cancer therapy and has broad applicability to other systems and therapies as we gain this understanding. The proposal has high translational relevance as we are developing novel therapeutic combinations that may be tested in the clinic.